Ultrasound contrast agents (UCA) are typically gas-filled microbubbles that are administered intravenously to the systemic circulation. Microbubbles have a high degree of echogenicity, that is, they reflect the ultrasound waves. The echogenicity difference between the gas in the microbubbles and the soft tissue surroundings of the body is immense. Thus, ultrasonic imaging using microbubble contrast agents enhances the ultrasound backscatter, or reflection of the ultrasound waves, to produce a unique sonogram with increased contrast due to the high echogenicity difference. Contrast-enhanced ultrasound can be used to image blood perfusion in organs, measure blood flow rate in the heart and other organs, and has other applications as well.
A series of surfactant-based UCA, composed of sonicated mixtures of non-ionic surfactants which self-assemble around a gaseous core, have been developed (U.S. Pat. No. 5,352,436). One particular agent, ST68, consists of SPAN® 60 (sorbitan monostearate) and TWEEN® 80 (polyoxyethylene sorbitan monooleate) filled with octafluoropropane (a PFC gas) (Basude et al., 2001, Ultrasonics, 39, 437-44). This agent can consistently be produced with a mean size of 1.5 to 2 μm and produces over 20 dB enhancement for doses less than 100 μl/l in vitro (Basude et al., 2000, Ultrasound Med. Biol., 26, 621-8) and 0.05 ml/kg in vivo (Forsberg et al., 1996, 1996, IEEE Ultrasonics Symp., 2, 1337-40). However, further development of this agent is hampered by the fact that it consists of an aqueous suspension of bubbles, which have limited stability with time (less than a week at 4° C. An ideal UCA has been described as being stable at room temperature for at least 6 months (Wang et al., 1996, J. Phys. Chem., 100, 13815-21).
The technique of freeze-drying, or lyophilization, has been implemented to increase the shelf-life and stability of vaccines, viruses, and proteins in pharmaceutical production (Jennings, 1999, Lyophilization: Introduction and Basic Principles. Interpharm. Press. Denver, Colo.) and in liposomes as drug carriers with and without acoustic reflectivity (i.e. increased echogenicity) (Huang et al., 2002, Cell Molec. Biol. Letters, 7, 233-5; Hua et al., 2003, Drying Technol., 21, 1491-505). However, this process generates stresses during the freezing and drying stages which could destabilize the suspension and destroy the bubbles (Abdelwahed et al., 2006a, Euro. Pharm. Biopharm., 63, 87-94). Some agents, such as liposomes, require the addition of cryoprotectants to aid stability during freezing (Ozer et al., 1988, Acta Pharm. Technol., 34, 129-39) or lyoprotectants to help prevent structural and functional integrity loss that occurs during the drying process (Jennings 1999, supra). This is achieved by preventing fusion and aggregation during freeze-drying thus allowing for better reconstitution (Hua et al., 2003, supra). It has been suggested that the major damaging factors associated with freeze-drying liposomes are lipid-phase transition and fusion (Crowe et al., 1997, Cryobiol., 35, 20-30).
In order to be used safely in a clinical setting as well as to access various biological compartments, a contrast agent must have a diameter less than 8 μm. Typically, it has been difficult to fabricate ultrasound contrast agents in the nanometer range that are as functionally effective as their micrometer counterparts. In addition, all microbubble UCA suffer from a lack of stability, and being susceptible to degradation when freeze-thawed. Accordingly, there is an ongoing need in the art for the development of stable ultrasound contrast agents in the sub-micron size-range. There is also a need for an ultrasound contrast agent that is not susceptible to freeze-drying degradation. The present invention fills this need.